Structural and mechanistic studies of AAA+ proteinsAAA+ family proteins utilize energy from ATP hydrolysis to perform mechanical works during the process of DNA replication, in protein activation by remodeling, and in protein refolding and degradation, through which they play important roles in various cellular activities, including transcription regulation, membrane fusion, cell cycle control, and so on. AAA+ proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the sub-molecular level and would be beneficial to understanding functions of other ATP utilizing enzymes such as ABC transporters. In collaboration with Dr. M. Maurizi of LCB, my lab has been working on structural studies of the prokaryotic ClpA protein unfoldase, which is a component of the Clp proteases that are essential in many organisms and highly conserved. We determined the structure of full-length ClpA, the regulatory component of the ClpAP complex. We also determined the structures N-terminal domain of ClpA and its complex with ClpS, an adaptor protein that plays a role in selecting substrates for degradation. We analyzed the structures of the N-domain of ClpA and identified potential sites for its interaction with substrates. In collaboration with Dr. C. Li of NCI, my lab also crystallized the N-D1 fragments of a few human AAA+ protein p97 mutants, which are deficient in ubiquitin binding. We determined crystal structures of these mutants and found that the mutant proteins are all in a different conformation from previous reports and are bound with ATP, which was not observed for the D1 domain. We are furthering our effort to obtain crystals of N-D1mutants in different nucleotide binding states and testing biochemically binding affinities for ubiquitin under these nucleotide states.